SNARE-Mediated Membrane Fusion Probed Using a Synthetic Organelle in the Living Bacterium.

Studies on the mechanisms and regulation of functional assemblies of SNARE proteins mediating membrane fusion essentially make use of recombinant proteins and artificial phospholipid bilayers. We have developed an easy-to-use in vivo system reconstituting membrane fusion in living bacteria. It relies on the formation of caveolin-dependent intracytoplasmic cisternae followed by the controlled synthesis of members of the synaptic SNARE machinery. Only when a SNARE complex is formed with its intact components does the docking and subsequent fusion occur between the cisternae and the plasma membrane that is accompanied by the disappearance of the former. The phenotypic response of the bacterial cell to fusion events is a remarkable increase in cell body length due to an expansion of the plasma membrane. Therefore, such an easy-to-observe phenotype makes this system amenable to structure-function studies of SNAREs. We describe here the specific ways to produce caveolin and the SNARE proteins from compatible plasmids upon bacterial transformation and to obtain the elongated cell phenotype. We also provide protocols to carry out the preparation of cell culture samples suitable for biochemical and light microscopy analysis.